Receptor activator of nuclear factor-kappa ligand, OPG, and IGF-I expression during orthodontically induced inflammatory root resorption in the recombinant human growth hormone–treated rats
To investigate the effects of growth hormone (GH) on local receptor activator of nuclear factor-kappa ligand (RANKL), OPG, and IGF-I expression during orthodontically induced inflammatory root resorption in rats. Forty Wistar rats (gender: male; age: 7 weeks) were randomly divided into control and experimental groups. A force of 50 g was applied to move the right upper first molars mesially. The experimental and control groups received daily subcutaneous injections of recombinant human growth hormone (GH; 2 mg/kg) and equivalent volumes of saline, respectively. The rats were sacrificed on days 1, 3, 7, and 14. Micro–computed tomography–reconstructed images of the upper right first molars were used to survey root resorption and tooth movement. Horizontal sections of the maxillae were prepared for hematoxylin and eosin, tartrate-resistant acid phosphatase, and immunohistochemical staining. Resorption lacunae appeared on the compressed side of the distal buccal root of the right first molar on days 7 and 14. Compared with the control groups, GH-treated groups showed more RANKL-positive cells and osteoclasts on day 3 and more OPG- and IGF-I–positive cells and fewer odontoclasts on days 7 and 14. Indexes of root resorption were lower and tooth movement was faster in the GH-treated groups than in the control groups on days 7 and 14. The inhibitory effect of GH on root resorption by heavy force might be mediated by RANKL/OPG and IGF-I. Short-term GH administration may be a method with which to reduce root resorption and shorten treatment time, especially in patients who are susceptible to root resorption.ABSTRACT
Objective:
Materials and Methods:
Results:
Conclusions:
INTRODUCTION
Orthodontically induced inflammatory root resorption (OIIRR) is an unavoidable iatrogenic damage that occurs during orthodontic treatment.1 OIIRR is extremely complicated and is accompanied by periodontal tissue remodeling.1 The changed microenvironment induces inflammatory cells to migrate to periodontal tissue and to produce various inflammatory mediators that stimulate bone resorption and formation on the compressed and tension sides, respectively, through affecting cells in periodontal tissues.2,3 In general, cementum is less affected by osteoclastic resorption than is alveolar bone because of the unmineralized cementum, periodontal ligament (PDL), and cells, unless the force is excessive in magnitude or duration.4,5
The receptor activator of nuclear factor-kappa ligand (RANKL), a proinflammatory cytokine, is expressed in osteoblastic cells and induces osteoclast differentiation, activation, and survival by binding to RANK on osteoclast-like cell lineages.6 However, OPG, a decoy receptor for RANKL, blocks osteoclastogenesis and accelerates mature osteoclast apoptosis by inhibiting RANKL binding to RANK.6 The OPG/RANKL/RANK axis plays a crucial role in root resorption.7 The RANKL/RANK system is involved in root resorption in naturally exfoliated primary teeth and OIIRR.8,9 RANKL and OPG messenger RNA (mRNA) expression increased in tissues during root resorption by heavy force.10
Growth hormone (GH) is responsible for postnatal bone growth and bone mass maintenance. The effects of GH on bone metabolism were previously believed to be mediated mostly through IGF-I.11 However, evidence indicates that the OPG/RANKL/RANK axis is also involved in the regulation of the effects of GH on bone cells.12–14 OPG secretion and mRNA expression in human osteoblast–like cells increased with GH treatment,12 and OPG levels both in serum and in cortical and trabecular bone explants from GH-deficient (GHD) patients were enhanced by GH treatment.13,14
The GH-OIIRR relationship remains unknown. GH might affect OIIRR through RANKL/OPG and IGF-I. Therefore, the purpose of this study was to investigate the effect of recombinant human GH (rHGH) on root resorption as well as on RANKL, OPG, and IGF-I syntheses on the compressed sides in rats.
MATERIALS AND METHODS
Animals and rHGH Treatment
Forty Wistar rats (gender: male; age: 7 weeks; weight: 215 ± 16 g) were obtained from the Experimental Animal Centre of Hubei Province. The animal experimental protocol was approved by the Wuhan University School and Hospital of Stomatology Ethics Committee, and the guidelines for laboratory animal care were followed throughout this study.
The animals were randomly divided into control (n = 20) and experimental (n = 20) groups. They were allowed to acclimate for 1 week. The experimental group received daily subcutaneous injections of rHGH (Somatropin, United Biotech, Shanghai, China) with a total dose of 2 mg/kg/d, twice daily in equal doses.15,16 The control group received twice-daily subcutaneous injections of saline in weight-based volumes equivalent to the rats in the experimental group. The initial injection was administered when the orthodontic appliance was bonded to the rat's teeth, and the injection site was changed frequently to prevent subcutaneous adhesion.
Rat Model of OIIRR
The method described by Gonzales et al.5 was used, with a slight modification. In brief, the rats were anesthetized with intraperitoneally administered 8% chloral hydrate (5 mL/kg body weight). The cervical portion of the maxillary incisors was prepared by grinding of shallow grooves using a diamond bur. The incisors were then treated with self-etching bonding material (GC, Tokyo, Japan) for 60 seconds. Sentalloy closed coil springs (TOMY, Tokyo, Japan) that delivered 50 g of force5,9 were ligated between the maxillary incisors and the maxillary right first molars. Composite resin was then placed over the surfaces of the incisors to prevent ligatures from loosening (Figure 1a). The mandibular incisors were ground at the beginning of the study and on day 6 to decrease occlusal interference. The molar on the left side was used as an appliance-free control.
Citation: The Angle Orthodontist 85, 4; 10.2319/052014-361.1
Micro–Computed Tomography
On days 1, 3, 7, and 14, five rats from two groups, respectively, were sacrificed, and the maxillae were dissected and fixed in 4% paraformaldehyde for 48 hours. The samples were then fixed in a tube and scanned by micro–computed tomography (Micro-CT; nano–computed tomography; Scanco Medical AG, Bruttisellen, Switzerland). The following scanning parameters were used: voltage, 50 kV; electrical current, 0.1 mA; magnification, 10×; pixel size, 1024 × 1024; and slice thickness, 10 µm. The total scanning time for each specimen was 1 hour. The cross-sectional data were exported in TIFF format and then imported to Mimics 10.0 (Materialise Software, Leuven, Belgium) for the reconstruction of the morphology of the distal buccal root, root resorption craters, and crown of each specimen.
Reconstructed images were used to confirm root resorption and to measure the volume of root resorption craters and orthodontic tooth movement (OTM), respectively. Root resorption craters on the distal surfaces and the apical thirds of the roots were not evaluated because craters on the distal surfaces were scarcely detectable, and anatomic variations in the apical region led to difficulties in defining the craters. The volume of the craters on the cervical two-thirds of the mesial surface of the distal buccal root of the right maxillary first molar (M1) and the cervical two-thirds of the root were measured, respectively, and their ratio was calculated as the index of root resorption (RRI). Distance between the midpoint of the distal surface of M1 and that of the mesial surface of the maxillary second molar (M2), which paralleled the occlusal surface, was defined as the OTM of M1. An author who was blinded to the grouping performed the measurements.
Histological Analysis
After micro-CT, the tissues were demineralized in 10% ethylene diamine tetraacetic acid (pH 7.2–7.4) at room temperature for 6 weeks, dehydrated, and embedded in paraffin. Continuous slices (3 µm) in the horizontal direction were prepared for hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and immunohistochemical staining for RANKL, OPG, and IGF-I. PDL in the cervical two-thirds of the mesial surface of the distal buccal root of M1 was studied.
TRAP Staining
Osteoclastic activity was demonstrated using the TRAP kit (Sigma, Saint Louis, MO, USA). The sections were dewaxed, rehydrated, and incubated in TRAP reagent, which was prepared according to the manufacturer's instructions, for 1 hour at 37°C. They were then stained with hematoxylin. A section incubated in substrate-free medium was regarded as the TRAP control. Multinuclear TRAP-positive cells on the surface of alveolar bone and root were osteoclasts and odontoclasts, respectively.
Immunohistochemistry
The sections were deparaffinized in xylene, rehydrated, and treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Polyclonal goat anti-RANKL antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal goat anti-OPG antibody (1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and polyclonal rabbit anti–IGF-I antibody (1:250, Proteintech, Chicago, IL, USA) were used as primary antibodies overnight at 4°C. The specimens were then washed and incubated with the Polink-2 plus polymer HRP detection system (ZSGB-BIO, Beijing, China), according to the manufacturer's instructions. After being washed with phosphate-buffered saline three times for 10 minutes, the specimens were made chromogenic using diaminobenzidine and were counterstained with hematoxylin. Some sections, incubated with phosphate-buffered saline instead of the primary antibodies, were used as immunohistochemical controls.
Cell Counting and Statistical Analysis
Five randomly selected regions, viewed under 400× magnifications in the cervical two-thirds of the mesial surface of the distal buccal root from each specimen, were used to count multinuclear TRAP-positive osteoclasts and odontoclasts and RANKL-, OPG-, and IGF-I–positive PDL cells.
Data from each group were analyzed by the third author using SPSS 13.0 software (SPSS Inc, Chicago, Ill). This author was also blinded to the treatment. Differences in the means between the two groups at the same observation time were analyzed using the Student's t-test. The significance level was set at P < .05.
RESULTS
Root Resorption and Tooth Movement Based on Micro-CT Results
While resorption lacuna and tooth movement were not observed on the unloaded tooth (Figure 1d,f), the loaded tooth showed these features on days 7 and 14 (Figure 1e,g,h). The RRI was significantly lower and the extent of tooth movement was significantly greater in the experimental group than in the control group on days 7 and 14 (Figure 1i,j; Table 1).
H&E Staining
Fibers and cells in PDLs without orthodontic forces were in normal relation, and no resorption lacunae were observed (Figure 1b). By contrast, PDLs in the mesial area of the loaded roots were compressed and showed disorganized fibers and cells (Figure 1c). Multinucleate odontoclasts appeared in the root resorption lacunae after day 3.
TRAP Staining
Compared with the control group, TRAP-positive osteoclasts significantly increased on day 3 and decreased on day 7, and TRAP-positive odontoclasts significantly decreased on days 7 and 14 in the experimental group (Figure 2; Table 1).
Citation: The Angle Orthodontist 85, 4; 10.2319/052014-361.1
Immunohistochemical Observations
Compared with corresponding values in the control group, significantly more RANKL-positive PDL cells with higher RANKL/OPG ratios on day 3 and more IGF-I– and OPG-positive PDL cells with lower RANKL/OPG ratios on days 7 and 14 were observed in the experimental group (Figures 3 and 4; Table 2).
Citation: The Angle Orthodontist 85, 4; 10.2319/052014-361.1
Citation: The Angle Orthodontist 85, 4; 10.2319/052014-361.1
DISCUSSION
Using a previous method5 with slight modifications, we successfully established animal models of OIIRR by applying a heavy force of 50 g on the maxillary right M1 of rats. Micro-CT reconstructed images and H&E staining confirmed obvious resorption craters on the pressure sides of the distal buccal root on days 7 and 14. The present study showed that root resorption craters in the GH-treated rats were less than in the control group on days 7 and 14, indicating an inhibitory effect of GH on root resorption by heavy force.
RANKL/OPG and IGF-I present important functions in root resorption formation and repair.7–10,17,18 GH is able to regulate bone cells through the RANKL/OPG axis12–14 as well as by circulating or locally produced IGF-I.11 Hence, GH might influence root resorption by RANKL/OPG and IGF-I. This study showed that RANKL expression increased on day 3, while OPG and IGF-I expression increased on days 7 and 14 in GH-treated rats; this result indicated the stimulatory effects of GH on RANKL, OPG, and IGF-I expression in PDL cells over different OIIRR stages. RANKL upregulation by GH in the early phase of OIIRR may be mediated by inflammatory mediators. GH binding to GHR promotes the mitogen-activated protein kinase pathway19 and increases the production of proinflammatory mediators,20 such as tumor necrosis factor–α, interferon-γ, COX-2, and PGE2, which are involved in the acute inflammatory response during OIIRR and stimulate RANKL expression in PDL cells.3 The upregulatory effects of GH on OPG and IGF-I expression in PDL cells were not detected until day 7, likely because of the inhibition of OPG and IGF-I expression by compression forces17,21 and the insufficient concentration of GH in PDL to exert anabolic effect.
The ratio of RANKL/OPG determines the orientation of bone remodeling and the degree of root resorption.7 Higher RANKL/OPG ratio and more osteoclasts in the GH-treated rats on day 3 indicated that GH promoted bone resorption at the early stage of tooth movement, while lower RANKL/OPG ratios and fewer osteoclasts and odontoclasts in the GH-treated rats on days 7 and 14 demonstrated GH-accelerated bone formation and inhibited root resorption in later tooth movement stages. Consequently, GH quickened the rate of bone remodeling, which was consistent with the findings of previous studies.11,22 The rate of bone remodeling is known to affect tooth movement. High bone turnover accelerates tooth movement, whereas low bone turnover delays tooth movement.23,24 That is why GH increased the amount of tooth movement on days 7 and 14 in this study.
In this study, we used much higher and more frequent GH dosages than are normally required for GHD patients to overcome the immune response of rats to HGH. For this reason, determining whether similar consequences would be observed in patients receiving GH treatment concurrent with orthodontic treatment is a matter of concern. Further in vivo and in vitro experiments should be undertaken to verify the effects of GH on OIIRR.
CONCLUSIONS
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GH may inhibit root resorption by decreasing the ratio of RANKL/OPG and upregulating IGF-I expression.
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Short-term GH administration may be a method with which to reduce root resorption and shorten treatment time, especially in patients who are susceptible to root resorption.
(a) Intraoral photograph of the appliance. (b, c) H&E staining of unloaded root (b) and loaded root (c) on day 7. (magnification, 200×). D indicates dentine; PDL, periodontal ligament; and AB, alveolar bone. (d–h) Reconstructed three-dimensional micro-CT image showing root resorption and the distance of tooth movement on day 14; (d) unloaded root; (e) Loaded root; (f) No tooth movement in the appliance-free tooth; (g) Tooth movement in the control group; (h) Tooth movement in the experimental group; (i, j) Quantification of the ratio of the volume of root resorption craters (j) and tooth movement (i). * P < .05 compared with the control group. Arrow indicates root resorption lacuna.
TRAP staining (magnification, 400×). On day 3, the osteoclasts (arrows) on the alveolar bone surface were more numerous in the experimental group than in the control group (d, a, g). TRAP-positive multinucleate odontoclasts (arrows) appeared on the root resorption lacunae on days 7 (b, e) and 14 (c, f), with fewer in the experimental group than in the control group (h). D indicates dentine; PDL, periodontal ligament; and AB, alveolar bone. * P < .05 and ** P < .01 compared with the control group.
Immunohistochemical observations of RANKL and OPG expression in PDL cells (magnification, 400×). RANKL-positive cells (arrows) in the control (a) and GH-treated (b) rats on day 3; OPG-positive cells (arrows) in the control and GH-treated rats on days 7 (c, d) and 14 (e, f). RANKL-positive cells on day 3 (g) and OPG-positive cells on days 7 and 14 (h) were significantly increased in the experimental group. The RANKL/OPG ratio was greater in the experimental group than in the control group on day 3, but lower in the experimental group than in the control group on days 7 and 14 (i). D indicates dentine; PDL, periodontal ligament. * P < .05 and ** P < .01 compared with the control group.
Immunohistochemical observations of IGF-I expression in PDL cells (magnification, 400×). IGF-I–positive cells (arrows) were significantly more numerous in the experimental group (b, d) than in the control group (a, c) on days 7 and 14 (e). D indicates dentine; PDL, periodontal ligament. * P < .05 and ** P < .01 compared with the control group.
Contributor Notes